Dluccage biosensor for detecting fumonisin b1 and preparation method and application thereof

Abstract

The present application relates to a kind of biological sensor for detecting fumonisin B1 and its preparation method and application, the biological sensor includes dlucCage protein and lucKey protein;The amino acid sequence of the dlucCage protein is as shown in SEQ ID NO:1, or as the polypeptide with one, two, or three amino acid mutations to serine on sequence SEQ ID NO:1;The amino acid sequence of the lucKey protein is as shown in SEQ ID NO:2, or as the polypeptide with one, two, or three amino acid mutations on sequence SEQ ID NO:2.The biological sensor of the present application creatively realizes the good cooperation of antigen mimic epitope and dlucCage protein allosteric function, the connecting peptide of molecular head-to-tail extension design can stably connect larger size target binding motif, effectively break through the space limitation problem of traditional lucCage protein bolt domain to target binding motif grafting, with high sensitivity, strong specificity, signal readout fast and the like advantages, has good application prospect in the field of food safety detection.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and relates to harmful substance detection, in particular to a dlucCage biosensor for detecting fumonisin B1 and a preparation method and application thereof. BACKGROUND

[0002] Fumonisins (FBs) are a class of mycotoxins mainly produced by Fusarium moniliforme, which mainly pollute corn and other food crops. Fumonisins have significant toxicity to animal and human health, can cause porcine pulmonary edema, equine leukomalacia, avian diarrhea, liver and kidney damage in various animals, and are associated with human esophageal cancer and gastrointestinal diseases. Among them, fumonisin B1 (FB1) is the most common and toxic analogue, accounting for more than 60% of fumonisin pollution. FB1 has hazards such as neurotoxicity, teratogenicity, genetic toxicity, carcinogenicity, hepatotoxicity, immunosuppression and reproductive toxicity. Therefore, it is of great significance to establish a fumonisin B1 detection technology suitable for the whole food chain and capable of real-time analysis to ensure food safety.

[0003] Traditional methods for detecting fumonisin B1 include rapid screening methods and quantitative methods. Rapid screening methods are difficult to quantify, including the most widely used enzyme-linked immunosorbent assay, which has the disadvantage of cross-reaction with other structurally similar substances (such as FB2, FB3), and has slightly poor specificity. It is easily disturbed by the matrix and requires precise sample pretreatment. The immunochromatographic test strip method is prone to false positives or false negatives. Quantitative methods mainly include high-performance liquid chromatography (HPLC) and the currently recognized most authoritative confirmatory method of liquid chromatography-mass spectrometry (LC-MS / MS). LC-MS / MS has the advantages of ultra-high sensitivity and simultaneous detection of multiple toxins, but the instrument is very expensive, the operation and maintenance cost is high; the operation is complex and requires top-notch professional technicians; the sample pretreatment requirement is extremely high.

[0004] Allosteric proteins refer to a class of proteins whose biological activity is regulated by the non-covalent binding of specific molecules (called effectors or regulators), thereby causing changes in the three-dimensional conformation of the protein. This regulatory effect usually occurs at another site on the protein other than its active site, i.e., the allosteric site. According to the results after the effector binds, it can be divided into allosteric activation, positive synergistic effect, after the effector binds, the activity of the protein is enhanced. Allosteric inhibition, i.e., negative synergistic effect: after the effector binds, the activity of the protein is reduced. Allosteric proteins are used as biosensor elements, which have both the specificity of detecting targets (antigens / antibodies) and the conformational conversion characteristics of allosteric proteins. SUMMARY

[0005] Based on this, the purpose of the present application is to provide a dlucCage biosensor for detecting fumonisin B1 and application thereof.

[0006] The first aspect of the present application is to provide a dlucCage biosensor for detecting fumonisin B1, which comprises a dlucCage protein and a lucKey protein, wherein the amino acid sequence of the dlucCage protein is shown in SEQ ID NO: 1, or is a polypeptide with one, two, or three amino acid mutations on the sequence of SEQ ID NO: 1.

[0007] The amino acid sequence of the lucKey protein is shown in SEQ ID NO: 2, or is a polypeptide with one, two, or three amino acid mutations on the sequence of SEQ ID NO: 2.

[0008] In some embodiments, the amino acid mutation is serine (Serine).

[0009] In some embodiments, in the dlucCage protein, the polypeptide with amino acid mutations comprises at least one of the following: the 366th amino acid is mutated to serine (Serine) from the N-terminal calculation, the 380th amino acid is mutated to serine (Serine), and the 387th amino acid is mutated to serine (Serine).

[0010] In some embodiments, in the lucKey protein, the polypeptide with amino acid mutations comprises at least one of the following: the 220th amino acid is mutated to serine (Serine) from the N-terminal calculation, the 230th amino acid is mutated to serine (Serine), and the 238th amino acid is mutated to serine (Serine).

[0011] In some preferred embodiments, the amino acid sequence of the dlucCage protein is shown in SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 8.

[0012] In some preferred embodiments, the amino acid sequence of the dlucCage protein is shown in SEQ ID NO: 6, or the nucleotide sequence of the encoding gene of the dlucCage protein is shown in SEQ ID NO: 7.

[0013] In some preferred embodiments, the amino acid sequence of the lucKey protein is shown in SEQ ID NO: 9, or the nucleotide sequence of the encoding gene of the lucKey protein is shown in SEQ ID NO: 10.

[0014] In a second aspect of the present application, a recombinant expression vector or genetically engineered bacteria for expressing the dlucCage protein is provided in a biosensor for detecting fumonisin B1, wherein the recombinant expression vector or genetically engineered bacteria is inserted with a coding gene of the dlucCage protein in any of the above-mentioned biosensors; and / or a recombinant expression vector or genetically engineered bacteria for expressing the lucKey protein is provided, wherein the recombinant expression vector or genetically engineered bacteria is inserted with a coding gene of the lucKey protein in any of the above-mentioned biosensors.

[0015] In a third aspect of the present application, a preparation method of the biosensor is provided, which comprises the following steps:

[0016] S1. Constructing a recombinant expression vector for expressing the dlucCage protein and a recombinant expression vector for expressing the lucKey protein, respectively;

[0017] S2. Transforming the two recombinant expression vectors into different genetically engineered bacteria, respectively, to obtain two recombinant engineered bacteria for expressing the two proteins;

[0018] S3. Inducing the recombinant engineered bacteria to express the dlucCage protein, and purifying the dlucCage protein;

[0019] S4. Inducing the recombinant engineered bacteria to express the lucKey protein, and collecting the inclusion bodies, denaturing, renaturing and purifying the inclusion bodies to obtain the lucKey protein with biological activity.

[0020] In some embodiments, in step S4, the denaturation and renaturation of the inclusion bodies comprises: repeatedly washing the inclusion bodies with a washing buffer for 3-5 times until the supernatant is clear; dissolving the inclusion bodies with a denaturation lysis solution, and centrifuging to collect the supernatant; purifying the supernatant, and then performing stage gradient dialysis renaturation; the stage gradient dialysis renaturation comprises dialyzing in a renaturation buffer I for 12-16 hours, and then dialyzing in a renaturation buffer II for 12-16 hours; preferably, the renaturation buffer I comprises: pH 8.5, 20 mM Tris-HCl, 1 mM EDTA, 50 mM urea, 2.2 mM reduced glutathione and 5% (volume percentage) glycerol; and / or the renaturation buffer II comprises: pH 8.5, 20 mM Tris-HCl, 1 mM EDTA and 50 mM urea.

[0021] In a third aspect of the present application, a method for detecting fumonisin B1 is provided, which uses any of the above-mentioned dlucCage biosensors, and comprises the following steps:

[0022] S1. Adding an incubation buffer into a reaction well;

[0023] S2. Add the dlucCage protein and fumonisin B1 antibody into the biosensor, and mix well;

[0024] S3. Add the fumonisin B1 antibody, mix well, and the molar ratio of the dlucCage protein to the lucKey protein is 1:(1-3);

[0025] S4. Add the sample to be tested, mix well, and incubate;

[0026] S5. Add furimazine substrate, and measure the luminescence intensity.

[0027] In some embodiments, in step S4, incubation is performed at 36-38℃ for 50-70 minutes.

[0028] In some embodiments, the molar ratio of the dlucCage protein to the lucKey protein is 1:(1-2), and more preferably 1:1.

[0029] In some embodiments, the incubation buffer comprises 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% Tween-20, 1 mg / ml BSA, and pH 7.4.

[0030] In some embodiments, the coding gene of the dlucCage protein is inserted into the Nde I and Xho I enzyme cutting sites of the pET-21b(+) vector to construct a recombinant expression vector for expressing the dlucCage protein.

[0031] In some embodiments, the coding gene of the lucKey protein is inserted into the Nde I and Xho I enzyme cutting sites of the pET-21b(+) vector to construct a recombinant expression vector for expressing the lucKey protein.

[0032] In some embodiments, the recombinant expression vector can be an E. coli expression vector, including the pET series, such as pET-21b(+).

[0033] In some embodiments, the genetically engineered bacteria are E. coli, such as Lemo21(DE3) strain.

[0034] The dlucCageFB1 protein biosensor has the advantage of significantly widening the target applicability, which can stably connect larger size target binding motifs through the linker designed at the head and tail of the molecule, effectively breaking through the spatial limitation problem of the target binding motif grafting of the traditional lucCage protein.

[0035] The dlucCage biosensor in the application is composed of an optimized dlucCage (dlucCageFB1_S) and a lucKey protein, wherein the N terminal of the cage domain and the C terminal of the latch domain in the dlucCage are connected with a mimic epitope polypeptide of fumonisin B1 antibody through specific connecting peptides, and the latch domain is integrated with a split luciferase SmBit fragment. The application utilizes the bivalent binding characteristics of the fumonisin B1 antibody to regulate the configuration change of the sensor: when the target fumonisin B1 exists, it competitively blocks the binding of the antibody and the mimic epitope, releases the restraint of the latch domain, and makes the lucKey protein trigger the luciferase complementary reaction through competitive binding, thereby generating signal output. The sensor innovatively realizes the synergistic effect of the antigen mimic epitope and the allosteric function of the dlucCage protein, has the advantages of simple preparation, low cost, high sensitivity, strong specificity, rapid signal reading and the like, and has a good application prospect in the field of food safety detection. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 It is a schematic diagram of the detection principle of the biosensor of the application.

[0037] Figure 2 It is an SDS-PAGE gel identification diagram of the dlucCage protein (A) and the lucKey protein (B) in the biosensor of the application.

[0038] Figure 3 It is a response diagram of the sensor to different concentrations of fumonisin B1 antibody when the concentration ratio of dlucCage:lucKey is 50nM:50nM.

[0039] Figure 4A It is a biosensor schematic diagram of dlucCage with different linker lengths.

[0040] Figure 4B It is a detection response effect diagram of dlucCage with different linker lengths.

[0041] Figure 4C It is a response effect diagram of dlucCageFB1_S:lucKey protein at different ratios.

[0042] Figure 5 It is a response effect diagram of dlucCageFB1_S protein and lucKey protein mutants with different affinities to fumonisin B1 antibody

[0043] Figure 6 It is a standard curve diagram based on the inhibition effect of fumonisin B1 standard product when dlucCage:lucKey is 50nM:50nM.

[0044] Figure 7 Specificity detection chart for the biosensor described in Example 6 of the present application. DETAILED DESCRIPTION

[0045] For the purposes of the present application, a more complete description of the application will be provided below. The present application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that the disclosure of the present application can be more thoroughly and completely understood.

[0046] The experimental methods in the following examples, unless otherwise specified, are generally performed according to routine conditions, for example, in the fourth edition of Molecular Cloning: A Laboratory Manual, edited by Green and Sambrook, which was published in 2013, or according to the conditions recommended by the manufacturer. The various common chemical reagents used in the examples are commercially available products.

[0047] Unless otherwise defined, all technical and scientific terms used in the present application have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used in this application, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0048] Reference is made to Figure 1 In some embodiments of the present application, a dlucCage biosensor for detecting fumonisin B1 includes a dlucCage protein having an amino acid sequence as set forth in SEQ ID NO: 1, or a polypeptide having one, two, or three amino acid mutations in the sequence of SEQ ID NO: 1, and a lucKey protein having an amino acid sequence as set forth in SEQ ID NO: 2, or a polypeptide having one, two, or three amino acid mutations in the sequence of SEQ ID NO: 2.

[0049] The dlucCage protein comprises a cage domain and a padlock domain; wherein the cage domain comprises a cage domain body and a fumonisin B1 polypeptide mimetic epitope; the cage domain body is connected to the fumonisin B1 polypeptide mimetic epitope through a specific linker, the fumonisin B1 polypeptide mimetic epitope is arranged at the N-terminus of the cage domain and the C-terminus of the padlock domain, and the padlock domain integrates a split luciferase SmBit fragment. The lucKey protein comprises a polypeptide that strongly binds to the cage domain of the dlucCage protein and a split luciferase LgBit fragment. The present application utilizes the bivalent binding property of the fumonisin B1 antibody to regulate the configuration change of the biosensor: when the target fumonisin B1 exists, it competitively blocks the binding of the antibody to the mimetic epitope, releases the padlock domain from the constraint, and triggers the luciferase complementary reaction through competitive binding to produce signal output.

[0050] In some embodiments of the present application, the preparation method of the dlucCage protein in the biosensor is also disclosed, comprising:

[0051] (a) inserting the nucleotide sequence shown in SEQ ID NO: 11 into the Nde I and Xho I enzyme cutting sites of the pET-21b(+) vector to construct a recombinant expression vector;

[0052] (b) transforming the recombinant expression vector obtained in step (a) into the Escherichia coli Lemo21(DE3) strain to obtain a recombinant engineering bacteria;

[0053] (c) inducing the recombinant engineering bacteria to express the dlucCage protein;

[0054] (d) purifying the obtained dlucCage protein.

[0055] In some more embodiments, the steps (c) and (d) comprise: inoculating the positive recombinant engineering bacteria into LB liquid medium containing ampicillin and culturing at 37°C to the logarithmic growth phase; adding IPTG with a final concentration of 0.4 mM and inducing the culture at 18°C for 16 hours; centrifuging to collect the bacterial cells, resuspending in lysis buffer; ultrasonic crushing of the cells, centrifuging to collect the supernatant; loading the supernatant onto a nickel affinity chromatography column equilibrated, and sequentially performing washing and elution operations; collecting the eluate and concentrating, and storing the purified protein at -80°C.

[0056] In some embodiments, the lysis buffer comprises: 20 mM Tris-HCl (pH 8.0), 300 mM NaCl, 30 mM imidazole, 1 mM PMSF, and 0.02 mg / ml DNase.

[0057] In some embodiments, the washing buffer comprises: 20 mM Tris-HCl (pH 8.0), 300 mM NaCl, 30 mM imidazole.

[0058] In some embodiments, the elution buffer comprises: 20 mM Tris-HCl (pH 8.0), 300 mM NaCl, 300 mM imidazole.

[0059] Some embodiments of the present application also relate to a method for preparing lucKey protein in a biosensor, comprising:

[0060] (a) inserting the nucleotide sequence shown in SEQ ID NO: 12 between the Nde I and Xho I enzyme cutting sites of a pET-21b(+) vector to construct a recombinant expression vector;

[0061] (b) transforming the recombinant expression vector obtained in step (a) into an Escherichia coli Lemo21(DE3) strain to obtain a recombinant engineering bacterium;

[0062] (c) inducing the recombinant engineering bacterium to express lucKey protein and collecting inclusion bodies;

[0063] (d) denaturing, renaturing and purifying the inclusion bodies to obtain active lucKey protein.

[0064] In some more embodiments, steps (c) and (d) specifically comprise:

[0065] The positive recombinant engineering bacterium is inoculated into LB liquid medium containing ampicillin and cultured at 37°C until the logarithmic growth phase; 0.4 mM IPTG is added to a final concentration, and the culture is induced at 18°C for 16 hours; the bacterial cells are collected by centrifugation, resuspended in lysis buffer, and the cells are broken by ultrasonic treatment, and the precipitate is collected by centrifugation as inclusion bodies; the inclusion bodies are repeatedly washed with washing buffer for 3-5 times until the supernatant is clear; the inclusion bodies are dissolved with denaturing lysis solution, and the supernatant is collected by centrifugation; the supernatant is purified by nickel affinity chromatography column, and the operations of equilibration, sample loading, washing and elution are sequentially performed; the eluate containing lucKey protein is collected and renatured by stage gradient dialysis; the renatured protein is concentrated and stored at -80°C.

[0066] In some embodiments, the lysis buffer comprises: 20 mM Tris-HCl (pH 8.0), 300 mM NaCl, 30 mM imidazole, 1 mM PMSF and 0.02 mg / ml DNase.

[0067] In some embodiments, the denaturing lysis buffer comprises: 8 M urea, 20 mM Tris-HCl (pH 8.0), 2 mM imidazole, and 3 mM DTT.

[0068] In some embodiments, the denaturing lysis buffer comprises: 8 M urea, 20 mM Tris-HCl (pH 8.0), 2 mM imidazole, and 3 mM DTT.

[0069] In some embodiments, the denaturing lysis buffer comprises: 8 M urea, 20 mM Tris-HCl (pH 8.0), 2 mM imidazole, and 3 mM DTT.

[0070] In some embodiments, the denaturing lysis buffer comprises: 8 M urea, 20 mM Tris-HCl (pH 8.0), 2 mM imidazole, and 3 mM DTT.

[0071] In some embodiments, the stage gradient dialysis comprises:

[0072] In some embodiments, the stage gradient dialysis comprises:

[0073] In some embodiments, the stage gradient dialysis comprises:

[0074] The fumonisin B1 monoclonal antibody in the examples can be prepared by the following preparation method:

[0075] 1. Immunization of animals

[0076] Preparation of fumonisin B1-BSA: 1.1 mg BSA and 0.15 mg FB1 (FB1 standard, item number: sigma, F1147-1MG, specification: 1 mg) were dissolved in 0.45 mL 0.01 mol / L PBS (pH 7.4), 0.45 mL 0.5% glutaraldehyde solution was added dropwise, and stirred at room temperature for 2 h. 0.1 mL glycine solution (1 mol / L, pH 7.0) was added and stirred for 10 min. PBS dialysis was performed at 4°C. Three 6-8 week old female Balb / c mice were selected, fumonisin B1-BSA was mixed with Freund's adjuvant in the same volume as the immunogen, vortexed until the immunogen was completely emulsified, and the mice were injected subcutaneously in multiple points. The first immunization used Freund's complete adjuvant, the immunogen dose was 120 μg per mouse; the subsequent immunization used Freund's incomplete adjuvant, and the immunogen dose was gradually reduced, immunized once every 3 weeks, a total of 3 times. One week after the second immunization, the mouse tail blood was taken, and the serum antibody titer and inhibition rate in the tail blood were determined by indirect ELISA. Three days before fusion, the mouse with the highest titer was directly boosted with the immunogen, and the immunization dose was 40 μg per mouse.

[0077] 2. Preparation and screening of hybridoma cells

[0078] The selected immunized mice were sacrificed by the neck, the spleen was taken out on the clean bench, and the mouse myeloma cells SP2 / 0 were mixed with the spleen cells at a ratio of 1:10, and 50% PEG was added for cell fusion. Add dropwise to the 96-well cell culture plate coated with feeder cells. After fusion, the cell state was observed under a microscope, and semi-quantitative liquid exchange was performed on the 5th day after fusion, and full liquid exchange was continued to the 7th day.

[0079] The cells were fused for 7-10 days, the cell supernatant in the cell culture plate was aspirated, the secretion of antibodies in the cell supernatant was determined by indirect ELISA, and the positive cell plate wells were screened. The limited dilution method was used for subcloning. Coat fumonisin B1-OVA, PBS for blank control, culture medium for negative control, and serum of mouse eyeball blood after immunization for positive control. When the selected monoclonal cells have a positive rate of 100% in the 96-well cell culture plate, they are determined to be positive monoclonal, and they are timely frozen and expanded.

[0080] 3. Preparation and purification of monoclonal antibody ascites

[0081] Take 8-week-old Balb / c mice for pre-stimulation one week in advance, promote the secretion and aggregation of nutrients in the abdominal cavity of mice. The hybridoma cell strain obtained is expanded to the required number, centrifuged, carefully washed, resuspended with sterile 75% physiological saline, and then injected into the abdomen of mice by intraperitoneal injection. After the abdomen of the mouse is obviously swollen for about one week, the ascites is collected, centrifuged at 10,000 r / min for 10 min, and the supernatant is collected as the monoclonal antibody ascites.

[0082] The collected ascites is purified by Protein G affinity chromatography purification column. Before loading, the ascites is filtered by a microporous filter and used as the loading liquid. The Protein G column is equilibrated with the binding buffer (0.15M NaCl, 20mM Na2HPO4, pH 7.4), and the filtered ascites is loaded into the chromatography column. After equilibration, the elution buffer (0.1M citric acid, pH 2.5-3.0) is used for elution, and the antibody-rich eluate is collected. The eluate is acidic and should be immediately adjusted to neutral pH with neutralizing buffer (1M Tris-HCl, pH 9.0) to obtain fumonisin B1 monoclonal antibody (for specific sequence, see: Cai C, Xia Y, Guo Y, et al. Biosynthetic small molecule antigen mimics medicated lateral flow immunoassay for mycotoxin fumonisin B1 using nanobody fusion proteins [J]. Journal of Hazardous Materials, 2025, 487: 137194).

[0083] In the following sequence: Bold: Linker; Italic: FB1 mimotope; Underlined: SmBit dlucCageFB1_S amino acid sequence: SEQ ID NO: 1

[0084] dlucCageFB1_S nucleotide sequence: SEQ ID NO: 2

[0085]

[0086] Linker peptide in dlucCageFB1_M amino acid sequence SEQ ID NO: 3

[0087] GGSAEAAAKEAAAKEAGGSGGSGGS Linker peptide in dlucCageFB1_L amino acid sequence: SEQ ID NO: 4

[0088] GGSGGSAEAAAKEAAAKEAAAKEAGGSGGSGGS dlucCageFB1_S_L1: amino acid sequence: SEQ ID NO: 5

[0089] dlucCageFB1_S_L2 amino acid sequence: SEQ ID NO: 6

[0090] dlucCageFB1_S_L2 nucleotide sequence: SEQ ID NO: 7

[0091]

[0092] dlucCageFB1_S_L3 amino acid sequence: SEQ ID NO: 8

[0093] MGSHHHHHHGSGSENLYFQGNNAAMYSEMATDGGGGSGGGGSSKEAAKKLQDLNIELAR

[0094] KLLEASTKLQRLNIRLAEALLEAIARLQELNLELVYLAVELTDPKRIRDEIKEVKDKSKEIIRR

[0095] AEKEIDDAAKESKKILEEARKAIRDAAEESRKILEEGSGSGSDALDELQKLNLELAKLLLKA

[0096] IAETQDLNLRAAKAFLEAAAKLQELNIRAVELLVKLTDPATIRRALEHAKRRSKEIIDEAERA

[0097] IRAAKRESERIIEEARRLIEKAKEESERIIREGSGSGDPDIKKLQDLNIELARELLRAHAQLQR

[0098] LNLELLRELLRALAQLQELNLDLLRLASELTDPDEARKAIA VTGYRLFEEIL DSERLSREAAAASEKSSREAERSIREAAAASEKISREGGGGSGGGGSNNAAMYSEMATD*

[0099] lucKey amino acid sequence: SEQ ID NO: 9

[0100] MGSHHHHHHGSGSENLYFQGSGMVFTLEDFVGDWEQTAAYNLDQVLEQGGVSSLLQNLA

[0101] VSVTPIQRIVRSGENALKIDIHIIPYEGLSADQMAQIEEVFKVVYPVDDHHFKVILPYGTLVID

[0102] GVTPNMLNYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLITPDGSMLFRVTINSGGSGG

[0103] GGSGGGSGGSDEARKAIARVKRESKRIVEDAERLIREAAAASEKISREAERLIREAAAASEKISRE

[0104] lucKey nucleotide sequence: SEQ ID NO: 10

[0105] 5'-atgggtagccatcatcatcatcaccacggtagcggtagtgaaaatctgtattttcagggcagcggcatggtgtttaccctggaagattttgtgg gtgactgggaacagaccgcagcatataatctggatcaggtgctggaacagggtggcgttagtagcctgctgcagaatctggccgttagtgtgaccccgattcagcgtattgtgcgcagcggcgaaaatgccctgaaaattgatattcatatcatcccgtatgagggtctgagcgcagatcagatggcccagattgaagaagtttttaaagtggtttacccggtggatgatcatcattttaaagttattctgccgtatggtacactggttattgatggcgttaccccgaatatgctgaattattttggccgcccgtatgaaggcattgccgtttttgatggtaaaaagattaccgtgaccggtacactgtggaatggtaataagattattgatgaacgcctgattaccccggatggtagcatgctgtttcgcgttaccattaatagtggcggcagcggcggcggtggtagtggtggtggtagcggcggtagtgatgaagcacgcaaagccattgcacgtgtgaaacgcgaaagtaaacgcattgttgaagatgccgaacgcctgatccgcgaagcagcagccgccagtgaaaaaattagccgcgaagccgaacgcttaattcgtgaagcagcagcggcaagcgaaaaaattagtcgtgaataa-3'

[0106] MGSHHHHHHGSGSENLYFQGSGMVFTLEDFVGDWEQTAAYNLDQVLEQGGVSSLLQNLA

[0107] VSVTPIQRIVRSGENALKIDIHIIPYEGLSADQMAQIEEVFKVVYPVDDHHFKVILPYGTLVID

[0108] GVTPNMLNYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLITPDGSMLFRVTINSGGSGG

[0109] GGSGGGSGGSDEARKAIARVKRESKRIVEDAERLSREAAAASEKISREAERLIREAAAASEKISRE*

[0110] lucKey_k2 amino acid sequence: SEQ ID NO: 12

[0111] MGSHHHHHHGSGSENLYFQGSGMVFTLEDFVGDWEQTAAYNLDQVLEQGGVSSLLQNLA

[0112] VSVTPIQRIVRSGENALKIDIHIIPYEGLSADQMAQIEEVFKVVYPVDDHHFKVILPYGTLVID

[0113] GVTPNMLNYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLITPDGSMLFRVTINSGGSGG

[0114] GGSGGGSGGSDEARKAIARVKRESKRIVEDAERLSREAAAASEKSSREAERLIREAAAASEKISRE*

[0115] lucKey_k3 amino acid sequence: SEQ ID NO: 13

[0116] MGSHHHHHHGSGSENLYFQGSGMVFTLEDFVGDWEQTAAYNLDQVLEQGGVSSLLQNLA

[0117] VSVTPIQRIVRSGENALKIDIHIIPYEGLSADQMAQIEEVFKVVYPVDDHHFKVILPYGTLVID

[0118] GVTPNMLNYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLITPDGSMLFRVTINSGGSGG

[0119] GGSGGGSGGSDEARKAIARVKRESKRIVEDAERLSREAAAASEKSSREAERLSREAAAASE

[0120] KISRE*

[0121] ’

[0122] The application will be further described in detail below in connection with specific embodiments.

[0123] Prokaryotic expression and purification of dlucCage protein

[0124] Prokaryotic expression and purification of dlucCage protein, including the following steps:

[0125] The gene encoding the target protein (dlucCageFB1_S / dlucCageFB1_M / dlucCageFB1_L / dlucCageFB1_S_L1 / dlucCageFB1_S_L2 / dlucCageFB1_S_L3) protein was cloned into the Nde I and Xho I enzyme cutting sites between the pET-21b(+) vector, respectively, to construct a recombinant expression plasmid. The recombinant plasmid was transformed into E. coli Lemo21(DE3) competent cells, spread on LB plates containing ampicillin, and positive clones were selected.

[0126] A single colony was picked and inoculated into 5 ml of LB liquid medium containing ampicillin, and cultured at 37°C for 8 hours with shaking. The culture was transferred to 100 ml of LB liquid medium containing ampicillin at a ratio of 1:50, and cultured at 37°C for 4 hours with shaking. IPTG was added to a final concentration of 0.4 mM, and the culture was induced at 18°C for 16 hours. The bacterial cells were collected by centrifugation at 4000g for 15 minutes at 4°C. The bacterial cells were resuspended in 30 ml of lysis buffer (20 mM Tris-HCl pH 8.0, 300 mM NaCl, 30 mM imidazole, 1 mM PMSF, 0.02 mg / ml DNase). The cells were broken using an ultrasonic disrupter under ice bath conditions (5 seconds of work, 5 seconds of intermittent, total duration of 20 minutes). The lysate was centrifuged at 12000 rpm for 20 minutes at 4°C, and the supernatant was collected.

[0127] The supernatant was loaded onto a 1 ml HisTrap nickel affinity column previously equilibrated with a washing buffer (20 mM Tris-HCl pH 8.0, 300 mM NaCl, 30 mM imidazole). Two washes were performed with 10 column volumes (CV) of the washing buffer. The protein of interest was eluted with 8 CV of an elution buffer (20 mM Tris-HCl pH 8.0, 300 mM NaCl, 300 mM imidazole). The eluate was verified by SDS-PAGE gel electrophoresis (results not shown) and the correct eluate was concentrated using an Ultra-15 centrifugal filter unit and stored at -80°C after aliquoting. Figure 2

[0128] Preparation of lucKey proteins

[0129] The method for preparing lucKey proteins comprises the following steps:

[0130] The gene encoding the target protein (lucKey / lucKey_k1 / lucKey_k2 / lucKey_k3) was cloned into the Nde I and Xho I restriction sites of the pET-21b(+) vector, respectively, to construct a recombinant expression plasmid. The recombinant plasmid was transformed into E. coli BL21(DE3) competent cells.

[0131] The Lemo21(DE3) competent cells were spread on LB plates containing ampicillin and positive clones were selected.

[0132] ​The single colony was inoculated into 5 ml of LB liquid medium containing ampicillin and incubated at 37°C for 8 hours. The culture was transferred into 100 ml of LB liquid medium containing ampicillin at a ratio of 1:50 and incubated at 37°C for 4 hours. IPTG was added to a final concentration of 0.4 mM and the culture was induced at 18°C for 16 hours. The bacterial cells were collected by centrifugation at 4000 g for 15 minutes at 4°C. The bacterial cells were resuspended in 30 ml of lysis buffer (20 mM Tris-HCl pH 8.0, 300 mM NaCl, 30 mM imidazole, 1 mM PMSF, 0.02 mg / ml DNase). The cells were broken by using an ultrasonic disrupter under ice bath condition (working for 5 seconds, intermittent for 5 seconds, and total time of 20 minutes). After breaking, the solution was centrifuged at 12000 rpm for 20 minutes at 4°C, and the precipitate was repeatedly washed with washing buffer (20 mM Tris-HCl (pH 8.0), 0.5% (v / v) Triton X-100, 1 mM EDTA and 0.5 M NaCl) for 3-5 times until the supernatant was clear; the inclusion bodies were dissolved with denaturing lysis solution (8 M urea, 20 mM Tris-HCl (pH 8.0), 2 mM imidazole and 3 mM DTT), and the supernatant was collected by centrifugation; the supernatant was purified by nickel affinity chromatography column, equilibrated with equilibration buffer (8 M urea, 20 mM Tris-HCl (pH 8.0), 2 mM imidazole and 1 mM 2-mercaptoethanol), then loaded, washed and eluted with elution buffer (8 M urea, 20 mM Tris-HCl (pH 8.0), 500 mM imidazole and 1 mM 2-mercaptoethanol); the eluate containing lucKey protein was collected and then placed into a dialysis bag for dialysis in refolding buffer I (20 mM Tris-HCl (pH 8.5), 1 mM EDTA, 50 mM urea, 2.2 mM reduced glutathione and 5% (v / v) glycerol) for 12-16 hours; then the dialysis bag was placed into refolding buffer II (20 mM Tris-HCl (pH 8.5), 1 mM EDTA and 50 mM urea) for 12-16 hours, and then the dialysate was verified by SDS-PAGE gel electrophoresis (the results are shown in FIG. 3B), and the correct band eluate was concentrated by using an Ultra-15 centrifugal filter device, aliquoted and stored at -80°C. Figure 2

[0133] Example 3 Test of response characteristics of the sensor to fumonisin B1 antibody

[0134] ​Dilute dlucCageFB1_S and lucKey proteins to 500 nM. In each well of a white, opaque 96-well plate, pre-fill with 20 μL of incubation buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% Tween-20, 1 mg / ml BSA, pH 7.4). Then, add 10 μL of dlucCage protein and 10 μL of serially diluted fumonisin B1 antibody solution, mixing thoroughly. Next, add 10 μL of lucKey protein to each well and incubate at 37°C for 60 min. After incubation, add 50 μL of 15 μM furimazine substrate and measure the luminescence intensity. Results are as follows: Figure 3 As shown, the sensor exhibits a concentration-dependent gradient response to fumonisin B1 antibody. Based on this, the sensor architecture was subsequently modified and optimized.

[0135] Example 4: Optimization of dlucCage sensor linker length

[0136] To investigate the effect of linker length on the mimic epitope-antibody binding effect, we designed three linkers of different lengths to link the fumonisin B1 mimic epitope peptide, namely: short... (named dlucCageFB1_S), medium (named dlucCageFB1_M) and long (Named dlucCageFB1_L), meaning that except for the linker peptide sequence, it is identical to the corresponding component of dlucCageFB1_S. For example... Figure 4A As shown. The response of the three linker length designs to the fumonisin B1 antibody was then characterized, with the specific implementation steps being the same as in Example 3. Experimental results showed that only the short linker length design responded to the antibody; the other two did not. Figure 4B As shown. Therefore, the design with a short linker length (dlucCageFB1_S) was chosen as the target for subsequent experimental optimization.

[0137] The ratio of dlucCage sensor elements was also optimized, with the ratio of dlucCageFB1_S:lucKey protein elements set to 50nM:25nM, 50nM:50nM, 50nM:100nM, and 50nM:150nM. Experiments were then conducted following the steps in Example 3. The results showed that the best response signal to the target antibody was observed when the molar ratio of dlucCageFB1_S:lucKey protein elements in the sensor was 1:(1-3), and the highest signal gain was provided when the molar ratio was 1:1. Figure 4C ).

[0138] To fine-tune the binding affinity between lucKey protein and dlucCageFB1_S protein, we performed site-directed mutagenesis on specific hydrophobic amino acid residues involved in the interaction in the key peptide domains of both, replacing them with more hydrophilic serine (Serine, Ser, S). Specifically: in dlucCageFB1_S, from the N-terminus, the 366th alanine (Alanine, Ala, A), the 380th isoleucine (Isoleucine, Ile, I), and the 387th leucine (Leucine, Leu, L) were mutated to serine (Serine, Ser, S), respectively. The mutant with the 366th alanine (Alanine, Ala, A) mutated to serine (Serine, Ser, S) was named dlucCageFB1_S_L1; the mutant with the 366th alanine (Alanine, Ala, A) and the 380th isoleucine (Isoleucine, Ile, I) mutated to serine (Serine, Ser, S) was named dlucCageFB1_S_L2; the mutant with the 366th alanine (Alanine, Ala, A), the 380th isoleucine (Isoleucine, Ile, I), and the 387th leucine (Leucine, Leu, L) mutated to serine (Serine, Ser, S) was named dlucCageFB1_S_L3. In lucKey, from the N-terminus, the 220th, 230th, and 238th isoleucine (Isoleucine, Ile, I) were mutated to serine (Serine, Ser, S), respectively. The mutant with the 220th isoleucine (Isoleucine, Ile, I) mutated to serine (Serine, Ser, S) was named lucKey_k1; the mutant with the 220th and 230th isoleucine (Isoleucine, Ile, I) mutated to serine (Serine, Ser, S) was named lucKey_k2; the mutant with the 220th, 230th, and 238th isoleucine (Isoleucine, Ile, I) mutated to serine (Serine, Ser, S) was named lucKey_k3. By cumulative mutation, we constructed a series of dlucCageFB1_S_L1 / L2 / L3 and lucKey_k1 / k2 / k3 with decreasing binding affinities.

[0139] Subsequently, the luminescence response of each mutant combination in the presence of the target antibody was detected according to the experimental procedures of Example 3. The results showed (as shown in Figure 5 FIG. 2) that, Figure 5 FIG. 2) that,

[0140] The luminescence intensity of the combination mutant of dlucCageFB1_S_L2 and lucKey changed most significantly, and showed the highest response to the target antibody, so the dlucCageFB1_S_L2 protein (SEQ ID NO: 6) and the lucKey protein (SEQ ID NO: 9) were finally selected as elements of the biosensor for subsequent competitive detection of fumonisin B1.

[0141] Example 6

[0142] (1) Detection of the dlucCage biosensor for fumonisin B1 samples:

[0143] The dlucCageFB1_S_L2 protein and the lucKey protein were diluted to 500 nM, respectively. 10 μl of incubation buffer (10 mM Hepes, 150 mM NaCl, 3 mM EDTA, 0.005% Tween-20, 1 mg / ml BSA pH = 7.4) was added to each well of a white opaque 96-well plate, followed by 10 μl of the diluted dlucCageFB1_S_L2 protein. The fumonisin B1 antibody was diluted to 300 nM and added to the 96-well plate, 10 μl / well; then the fumonisin B1 standard solution was added in a dilution of 10 μl / well. Finally, the lucKey protein (10 μl / well) was added, and incubated at 37°C for 60 minutes. 50 μl of 15 μM furimazine substrate was added, and the luminescence intensity was measured. The standard curve (as shown in Figure 6 FIG. 3) was plotted with the logarithm of the concentration of the fumonisin B1 standard as the abscissa and the relative luminescence intensity as the ordinate. The results showed that when the sensor dlucCageFB1_S_L2: lucKey used a concentration of 50 nM: 50 nM, the half-inhibitory concentration (IC 50 ) of fumonisin B1 was 32.24 ng / ml, the detection limit was 7.027 ng / ml, and the linear range was 11.09-52.70 ng / ml.

[0144] (2) Specific detection of the sensor:

[0145] To evaluate the specificity of the biosensor of the present application, the interference of (dlucCageFB1_S_L2 protein and lucKey protein) on common mycotoxins such as ochratoxin A (OTA), deoxynivalenol (DON), zearalenone (ZEN), aflatoxin B1 (AFB1) and the like at a concentration of 1 μg / ml was tested. According to the above steps, various interference toxin solutions were added in the sample to be tested step (instead of fumonisin B1 standard), and the luminescence intensity was detected and recorded.

[0146] The results, as shown in Table 1, show that the sensor of the present application has high specificity for fumonisin B1, and other toxins have no obvious cross-reaction at a higher concentration. Figure 7

[0147] The above-described embodiments only express several embodiments of the present application, which are described in detail, but should not be construed as limiting the scope of the patent. It should be noted that for those skilled in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are all within the scope of protection of the present application. Therefore, the scope of protection of the patent of the present application should be subject to the appended claims.​

Claims

1. A dlucCage biosensor for detecting fumonisin B1, characterized in that, The biosensor comprises a dlucCage protein and a lucKey protein; wherein, The amino acid sequence of the dlucCage protein is shown in SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO:

8. The amino acid sequence of the lucKey protein is shown in SEQ ID NO:

9.

2. The dlucCage biosensor of claim 1, wherein, The amino acid sequence of the dlucCage protein is shown in SEQ ID NO: 6, or the nucleotide sequence of the coding gene of the dlucCage protein is shown in SEQ ID NO:

7.

3. The dlucCage biosensor of claim 1 or 2, wherein, The nucleotide sequence of the coding gene of the lucKey protein is shown in SEQ ID NO:

10.

4. The recombinant expression vector or genetically engineered bacteria for preparing the dlucCage biosensor for detecting fumonisin B1 according to claim 1, characterized in that, The recombinant expression vector or genetically engineered bacteria are used for expressing the dlucCage protein and the lucKey protein, respectively, and are recombinant expression vectors or genetically engineered bacteria into which the coding gene of the dlucCage protein and the coding gene of the lucKey protein are inserted.

5. The method of preparing the dlucCage biosensor according to any one of claims 1 to 3, characterized in that, The preparation method comprises the following steps: S1. Constructing recombinant expression vectors for expressing the dlucCage protein and the lucKey protein, respectively; S2. Transforming the two recombinant expression vectors into different genetically engineered bacteria, respectively, to obtain two recombinant engineered bacteria for expressing the two proteins; S3. Inducing the recombinant engineered bacteria to express the dlucCage protein, and purifying the dlucCage protein obtained; S4. Inducing the recombinant engineered bacteria to express the lucKey protein, collecting the inclusion bodies, denaturing, renaturing, and purifying the inclusion bodies to obtain the lucKey protein with biological activity.

6. The method of claim 5 wherein the step of forming the first and second layers comprises the step of: In step S4, the denaturation and renaturation of the inclusion bodies comprise: repeatedly washing the inclusion bodies with a washing buffer for 3-5 times until the supernatant is clear; dissolving the inclusion bodies with a denaturation lysis solution, and centrifuging to collect the supernatant; purifying the supernatant, and then performing stage gradient dialysis renaturation; the stage gradient dialysis renaturation comprises dialyzing in a renaturation buffer I for 12-16 hours, and then dialyzing in a renaturation buffer II for 12-16 hours; and / or the renaturation buffer II comprises: pH 8.5, 20 mM Tris-HCl, 1 mM EDTA, and 50 mM urea. ​ 7. The method of claim 6 wherein the step of forming the first and second layers comprises the step of: The renaturation buffer I comprises: pH 8.5, 20 mM Tris-HCl, 1 mM EDTA, 50 mM urea, 2.2 mM reduced glutathione, and 5% (volume percentage) glycerol. ​ 8. A method for detecting fumonisin B1, characterized by, The method comprises the following steps: S1. Adding an incubation buffer to a reaction well; S2. Adding the dlucCage protein and fumonisin B1 antibody in the biosensor of any one of claims 1-3, and mixing; S3. Adding the lucKey protein in the biosensor of any one of claims 1-3, and mixing, wherein the molar amount ratio of the dlucCage protein to the lucKey protein is 1: (1-3); S4. Adding a sample to be tested, and mixing and incubating; S5. Adding a furimazine substrate, and measuring the luminescence intensity.

9. The method of claim 8, wherein the fumonisin B1 is detected by, In step S4, incubation is performed at 36-38°C for 50-70 minutes; and / or The molar ratio of the use amount of the dlucCage protein and the lucKey protein is 1:1; and / or The incubation buffer comprises: 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% Tween-20, 1 mg / ml BSA, pH 7.4.

Images